Abstract Background: Venezuela is an endemic area for human and canine leishmaniosis due to Leishmania infantum and parasites of the Leishmania braziliensis and L. mexicana complexes. Limited data are available on feline leishmaniosis (FeL) in this region. The aim of this study was to describe clinical and diagnostic aspects of FeL in Venezuela. Results: Thirty-one domestic cats from urban areas of Lara State in Venezuela were enrolled. Twenty-five were healthy. Six other cats had solitary or multiple nodular lesions, which were located on the nose; ears; ears and nose; and nose, ears, tail and lower limbs. Cutaneous lesions were characterized by diffuse pyogranulomatous infiltrate in all sick cats with numerous intracellular and extracellular amastigotes, and immunohistochemistry was positive for Leishmania in five sick cats. All healthy cats were seronegative for L. infantum and L. braziliensis antigens by ELISA. Two out of five sick cats yielded a positive ELISA result to both Leishmania antigens with higher antibody levels to L. braziliensis compared to L. infantum. Significantly higher antibody levels by ELISA as well as a higher number of bands by Western blot (WB) were found for L. braziliensis when compared to L. infantum antigens in all sera from Venezuelan sick and healthy cats. All healthy cats were blood Leishmania spp. qPCR negative while three out of six sick cats were blood qPCR positive. All paraffin-embedded skin biopsies (n = 4) as well as cutaneous cytology (n = 3) were positive by Leishmania spp. qPCR in sick cats. Leishmania speciation was obtained only from the cutaneous lesion samples from cytological preparations of two out of three sick cats which were identified as infected with L. mexicana or a closely related specie. Conclusions: FeL should be included in the differential diagnosis list of nodular-ulcerative lesions. The most reliable diagnostic technique in sick cats is cytological or histopathological examination along with immunohistochemistry, since blood PCR and serology by ELISA might be negative. WB appears to be more sensitive in detecting infection. Cats with leishmaniosis from Venezuela are most likely infected with species of L. mexicana or a closely related species or the L. braziliensis species complex and not with L. infantum.